This invention relates to a method for calibrating a mass spectrometer. In particular, this invention relates to a method for calibrating a mass spectrometer using the mass spectrum of daughter or fragment ions produced by post-source decay of a meta-stable ion in a reflectron time-of-flight (TOF) mass spectrometer.
In a TOF mass spectrometer, meta-stable ions (also referred to as pre-cursor ions) are generated in an ion source from a sample and repelled from the source into a drift region. In the drift region, these meta-stable ions may break into fragments in a process known as post-source decay. Alternatively, post-source decay may be induced by laser or within a collision cell to produce fragment ions. These fragment or daughter ions are useful for determining the structure of the sample from which the meta-stable ions are generated. For example, in the case of a peptide sample, these daughter ions are related to the amino acid composition of the sample molecule and can therefore be used to deduce sequence information.
In this specification the terms parent ion, meta-stable ion and pre-cursor ion will be used interchangeably as will the terms daughter ion and fragment ion.
When analysing a sample by normal TOF mass spectrometry i.e. with or without a reflectron, the user is presented with data relating to the time that the ions have taken to travel through the drift region. The time taken is dependent on the mass to charge ratio of the ion. In order to convert the time of flight data into the more useful mass data, it is necessary to calibrate the mass spectrometer using a spectrum of a known compound in which the molecular identity and therefore the molecular weight of the ions observed is known. In this way it is possible to correlate flight time and molecular weight so that on analysing an unknown compound, it possible to assign weights to the unknown peaks on the basis of the flight time for the peak.
In a reflectron TOF mass spectrometer, the daughter ions formed in post-source decay are separated according to their velocity and according to their energy (which is related to their mass); whereas normal, parent ions all have approximately the same energy (having been accelerated by the same potential) and are separated according to their velocity only. Therefore the mass calibration for the daughter ions is not the same as for the normal (original meta-stable) ions.
Ions which undergo post source decay (PSD) do so (by definition) in the field free region. Thus ions that fragment in the source or the reflectron are not detected in the PSD fragment spectrum—either because they are selected out or do not reach the detector in time focus. Because there are no external fields (no external forces on the ions) momentum is conserved and all the fragments retain the velocity of the pre-cursor ion i.e., the velocity with which it left the ion source. The kinetic energy of the ions is given by the following equations:—
Pre-cursor ion:Ep = ½mpvp2Fragment ion:Ef = ½mfvp2(where Ep = Kinetic energy of precursor ion, Ef = kinetic energy of fragment ion, mp = mass of precursor ion, mf = mass of fragment ion and vp = velocity of precursor ion). 
Thus it follows that the ratio of the mass of a fragment ion to that of the pre-cursor is the same as the ratio of their kinetic energies:mf/mp=Ef/Ep
In a linear time-of-flight mass spectrometer we can see that because the velocities of the fragment and pre-cursor ions are the same there is no way of distinguishing between them—they arrive at the detector at the same time and therefore have the same measured mass.
In a reflectron time-of-flight mass spectrometer ions encounter a retarding field in the reflectron and travel into the reflectron to the point where their potential energy equals their kinetic energy. The ions are then turned around and reflected back out to emerge from the reflectron with the same speed but in the reverse direction. The reflectron is an energy analyser and can thus distinguish between pre-cursor ions and fragment ions and also fragment ions of different mass. This is the principle of fragment mass analysis in a reflectron time-of-flight mass spectrometer whatever type of reflectron is used. It applies to linear field reflectrons, where the voltage is stepped or scanned over multiple experiments in order to build up a complete fragment spectrum and also to curved field or quadratic field reflectrons which allow the fragment spectrum to be acquired in one shot.
The calibration of the time of flight spectrum for fragments is not the same as that of the pre-cursor ions. In the normal pre-cursor ion spectrum the ion energy is essentially the same for all mass whereas for the fragment ions there is a dependence of the ion energy on mass for the flight time in the reflectron. It is possible to calculate the calibration function for the fragment ions and relate this to the normal calibration function for the pre-cursor ions. Usually, the fragment mass calibration will depend on the ratio of the fragment mass with respect to the pre-cursor ion mass. However, for best mass accuracy and for practical reasons a calibration will be based typically on a fragment mass spectrum of a known compound. Typically a single known compound which gives rise to eight or so known fragments (of known masses) is used.
In the example of a curved field reflectron the basic calibration function has a form as follows. The actual mass, mact of the fragment ion can be related to the apparent mass, mapp that would be measured using the normal mass calibration (i.e., that of the pre-cursor ions). The ratio mact/mapp follows a curve which depends only on the ratio of mact to the pre-cursor mass, mpre. By knowing the mact for a standard compound and measuring the mapp the calibration curve can be defined for all pre-cursor masses. An example of such a curve is shown in FIG. 1. It can be seen from FIG. 1 that if the fragment has the same mass as the precursor ion, the apparent measured mass will be the same as the real mass. If however the fragment ion's actual mass is less than the precursor ion, the apparent measured mass (mapp) of the fragment ion will be greater than its actual mass (mact). In FIG. 1 the apparent mass of the fragment ion is approximately 1.4 times its actual mass when the actual fragment mass is 10% of the precursor ion mass. The exact shape of the calibration curve will be different for each spectrometer depending upon the reflectron and drift tube dimensions.
The inventors have realised that conventional methods of calibrating for PSD fragments in a reflectron mass spectrometer introduce errors into the calibration and lead to inaccurate mass measurement. This is due to a complication caused by the fact that the parent meta-stable ion has a natural isotope distribution, for example, from the natural abundance of carbon 13 isotopes in the molecule. The current invention provides a method of correcting for or avoiding these errors.
The errors and a method of correcting for or avoiding them are explained below.
Many atoms have more than one stable (non-radioactive) isotope, i.e., differing in the number of neutrons within the nucleus. The most common example is that of carbon 12C which has 6 protons and 6 neutrons giving a nominal mass of 12 Da but has a stable isotope with 7 neutrons, denoted 13C and a mass of 13Da. The 13C isotope has a natural abundance of 1.1% so that on average just over 1 in 100 carbon atoms is 13C. Similar behavior is seen for nitrogen, oxygen and sulphur. All of these atoms are present in significant quantities in organic molecules such as peptides and proteins so that the mass spectrum will show not one single peak but a distribution of peaks 1 Da apart according to the size of the molecule and the natural abundance of the isotopes of the atoms that make it up.
FIG. 2 shows the mass spectrum of the insulin b-chain. It can be seen that there are several peaks, each 1 Da (Dalton) apart due to the presence of isotopes in the insulin b-chain sample.
Similarly, fragment molecules also show isotope distributions. However the inventor has noticed that the separation of isotopic peaks in the fragment ion are not separated by 1 Dalton. The inventor has studied this phenomena and devised a method of spectrometer calibration and PSD fragment mass measurement which takes this into account and thus is more accurate than the prior art. This phenomena which has not previously been noticed, is described in more detail below.
The higher mass isotopes will be distributed randomly throughout the pre-cursor molecule and, in the absence of any unusual chemical effects, the higher mass isotopes will also be randomly distributed within the fragment molecule. When the fragmentation process occurs molecules with higher mass isotopes can therefore only form fragment ions with up to the same number of higher mass isotopes (but not more!).
In post-source decay this has a significant effect on the mass accuracy because fragments with the same number of higher mass isotopes (and therefore the same mass) can be produced by a pre-cursor with differing numbers of higher mass isotopes. For example, one parent ion will have a natural carbon 13 abundance and as this ion decays some daughter ions will contain only carbon 12 whilst other daughter ions will contain varying percentages of carbon 13.
FIG. 3 shows how fragments with the same number of higher mass isotopes can be produced by precursor ions with differing numbers of higher mass isotopes. In the interests of clarity FIG. 3 only considers the 13C carbon isotope which is the most significant isotope for organic compounds.
The top part of FIG. 3 shows the isotopic distribution of the parent ion, there are four peaks and each peak represents a parent ion with a different number of isotopes. The first peak 1 represents the mono-isotopic parent ion in which all of the carbon atoms are 12C atoms. The second peak 2 represents a parent ion containing only one 13C isotope. The third peak 3 represents a parent ion containing two 13C isotopes and the fourth peak 4 represents a parent ion containing three 13C isotopes. The peaks are equally spaced and 1 Dalton apart from each other, so as shown in FIG. 3 the mass of the first peak is Mp Daltons (where Mp is the mono-isotopic mass of the parent ion), the second peak mass is (Mp+1) Daltons, the third peak (Mp+2) Daltons and the fourth peak (Mp+3 Daltons).
The bottom part of FIG. 3 shows the isotopic distribution of a fragment ion originating from the precursor ion shown at the top of the FIG. 3. The distribution is shown by four peaks, again each peak represents a fragment ion containing a different number of 13C isotopes. The first peak 5 represents the mono-isotopic fragment ion which contains 12C atoms only and no isotopes, the second peak 6 represents a fragment ion which contains one 13C isotope only, the third peak 7 represents a fragment ion which contains two 13C isotopes and the fourth peak 8 represents a fragment ion which contains three 13C isotopes. The actual mass of the ion represented by the first peak 5 is Mf Daltons (Mf=the mono-isotopic mass of the fragment ion), the actual mass of the ion represented by the second peak 6 is (Mf+1) Daltons, (Mf+2) Daltons for the third peak 7 and (Mf+3) Daltons for the fourth peak 8. In a real mass spectrometer the measured masses and generated mass spectrum will be different as is explained later.
The arrows between the top and the bottom parts of FIG. 3 show the relationship between the isotopic distributions of the fragment and precursor ions. It shows which isotopic fragment ions can be produced by which isotopic precursor (parent) isotopic ions.
The mono-isotopic fragment ion 5 can be produced by any of the isotopic forms of the parent ion 1, 2, 3 or 4 as all of these will contain 12C atoms.
The first isotopic fragment ion 6 cannot be produced by the mono-isotopic parent ion (as the mono-isotope does not contain any 13C atoms), but can be produced by any one of the non-mono-isotopic parent ions 2, 3, or 4.
The second isotopic fragment ion 7 can be produced by any parent ion which contains at least two 13C atoms, i.e. by the second and third parent ion isotopes 3 and 4.
The third isotopic fragment ion 8 can only be produced by a parent ion having at least three 13C atoms, i.e. only by the third isotopic parent ion 4.
The measured mass of each fragment ion isotope will depend upon the parent isotope which it came from. As the ratio mact/mpre (the ratio of actual fragment ion mass to precursor ion mass) is different for each parent isotope, the calibration curve is slightly different and hence the measured mass will also be slightly different.
The difference in measured mass depends on the type of reflectron and the dimensions of the mass spectrometer but is finite for all instruments. It can be described as an offset in mass mo such that the difference between the actual and measured mass of the fragment ion is mo×n Daltons (Da) where, m0 is a mass offset parameter and n is extra mass (in Daltons) of the higher mass isotopic parent ion. (In the example of FIG. 3, n is the number of 13C atoms contained in the parent).
This mass offset effect can influence the mass measurement accuracy in two ways. Firstly, it leads to a broadening of the mass peak which effectively reduces mass resolution of the measurement. Secondly, the measured separation of the isotope peaks is not 1 Da but actually (1+mo) Da, where m0 is a parameter characterizing the mass offset. These effects are illustrated in FIG. 4 and FIGS. 5a and 5b. 
FIG. 4 shows this mass offset effect for the fragment ions resulting from a sample containing the parent ions 1 and 2 of FIG. 3.
The top part of FIG. 4 shows the mass spectrum which will be generated in the spectrometer by the parent ions. The first peak 10 is the mono-isotopic peak (generated by a parent ion 1 in which all the carbon atoms are 12C atoms) and the second peak 11 is the peak resulting from a parent ion 2 which has the same chemical formula as the parent ion 1, but in which one of the carbon atoms is a 13C atom.
The bottom part of FIG. 4 shows the peaks which will be generated in the spectrometer by the fragment ions. The first peak 20 is the mono-isotopic peak. The mono-isotopic peak is the peak generated by a mono-isotopic fragment ion which originated from a mono-isotopic parent ion. This relationship with the mono-isotopic parent ion is shown in FIG. 4 by an arrow pointing from the mono-isotopic parent peak 10 to the fragment ion's mono-isotopic peak 20.
The second peak 21 is the peak generated by a mono-isotopic fragment ion originating from a parent ion having one 13C atom amongst its carbon atoms. The actual mass of the fragment ion generating the peak 21 is the same as the actual mass of the fragment ion which generates the mono-isotopic peak 20, however its measured mass is greater because the ratio of the parent mass to the fragment is different.
The measured mass of the fragment ion which generates the mono-isotopic peak 20 is the same as its actual mass: Mf; the ratio of pre-cursor (parent) ion mass to actual fragment ion mass is Mp/Mf.
The actual mass of the fragment ion which generates the second peak 21 is also Mf, but its measured mass is Mf+m0; the ratio of pre-cursor to actual fragment mass for this fragment ion is Mp+1/Mf. As there are two peaks relating to the same actual mass fragment ion, the resolution of the spectrometer for fragment ions is reduced.
The third peak 22 shown at the bottom part of FIG. 4 is generated by a fragment ion containing one 13C isotope which originated from a parent ion containing one 13C isotope. The vertical dashed line in FIG. 4 shows the point 1 Dalton away from the mono-isotopic peak 21. It can be seen that due to the above described offset effect the spacing of the mono-isotopic peak 20 from the peak 22 is not 1 Dalton, but (1+m0) Daltons. The value of m0 depends upon other things on the type and size of the reflectron used.
This mass offset effect is a consequence of the fact that a fragment ion cannot have more higher mass isotopes than were in the pre-cursor ion that produced it. The effect is to shift the average of the mass distribution to higher mass by an amount depending on the abundance of higher mass isotopes in the pre-cursor ion and the size of mo.
While the offset effect has been described above with regard to the 13C isotope, it is not just carbon which produces this effect but also other isotopes such as nitrogen 15 and isotopes of oxygen and sulphur.
FIG. 5a is a mass spectrum showing the isotopic distribution of fragment ions without the mass offset effect (i.e. m0=0). FIG. 5b is a mass spectrum of the same fragment ions when the mass offset is m0=0.25. FIGS. 5a and 5b were generated by a computer model. It can be seen that the offset skews the shape of the mass spectrum towards the heavier masses.
While the above has been discussed in relation to a ‘mass offset’, it will be clear to a person skilled in the art that this could also be termed a ‘time of flight offset’ as mass need only be assigned to the various times of flight of the fragment ions at the end of the calibration process. The above discussion has assumed that the times of flight of the fragment ions are first converted to mass according to the parent ion calibration and then adjusted according to a calibration curve, e.g. such as that shown in FIG. 1. However it would also be possible to work in time of flight and to adjust the time of flight of the fragment ions with a similar calibration curve before finally assigning a mass at the end of the calibration process. However the above principles remain the same whether working in time of flight or mass.
It is possible to use a “smoothing” technique on the fragment mass isotopic distribution but this may lead to an error in the mass assignment as smoothing involves selection of a peak (usually the most abundant peak) and the centering of the distribution on this peak using an algorithm. In practice this smoothing leads to an averaging of the mass peaks in the distribution pattern, this average usually being distorted from the accurate mass by the higher mass isotope peaks within the distribution.
The following invention aims to ameliorate the above problems.